DESCRIPTION: The sphingomyelin pathway is a ubiquitous, evolutionarily conserved signaling system initiated by hydrolysis of the plasma membrane phospholipid sphingomyelin to generate ceramide. A number of direct targets for ceramide signaling have now been identified. Ceramide-activated protein (CAP) kinase is a membrane-bound, 97 kD Ser/Thr proline-directed protein kinase. Although the full range of activities of CAP kinase is as yet unknown, recent studies suggested it signals the proinflammatory action of TNFa via activating Raf-l. Dr. Kolesnick and his colleagues reported that CAP kinase complexes with, phosphorylates and stimulates Raf-1 in a ceramide- and TNF-dependent manner. While Raf-1 activation involves binding to GTP-ras, recent studies identified the existence of a kinase suppressor of ras (KSR). The investigators have identified KSR as CAP kinase. The present application extends these observations and examines the mechanism of signal transduction through KSR/CAP kinase. The specific aims of this application are: (1) The development of reagents for studying the extent of expression and biologic relevance of KSR/CAP kinase in mammalian cells. (2) The identification of KSR]CAP kinase domains involved in Raf-1 activation. (3) The characterization of proximal elements (ceramide, tyrosine kinases, endotoxin, ras) in KSR/CAP kinase activation. These studies will likely impact three distinct areas of cell biologic research. The demonstration of alternatives to the classic signaling systems for Raf-1 activation should provide a basis for new investigations into the role of Raf-1 in various cellular responses, including inflammation, proliferation and regulation of apoptosis. The availability of CAP kinase as a reagent should facilitate studies of mechanisms by which ceramide activates cellular targets to initiate transmembrane signaling. Lastly, the demonstration that KSR/CAP kinase may be integral to inflammatory signaling through the TNF receptor and should provide a foundation for studies into its role in the pathogenesis of TNF-mediated disease. It may also provide a biochemical target for pharmacologic manipulation of TNF action in vivo, with potential for clinical application.